The broad objectives of this proposal are to determine how centrosomes and kinetochores function and interact during mitosis to form the spindle, and how the forces for chromosome segregation are generated and regulated. Aim #1 involves the application of same-cell correlative video light microscopic (V-LM) and electron microscopic (EM) methods to investigate the mechanism of centriole/centrosome replication in sea urchin embryos in vivo, and in extracts of Spisula solidissima oocytes in vitro. Aim #2 is to evaluate how centrosomal microtubule (MT) behavior changes as the cell enters mitosis, and how exogenous tubulin affects MT behavior in vivo. For this study bovine MT protein, with or without a fluorescent label, will be injected into new lung cells (NLCs) and the cells followed by V-LM. Aim #3 is to quantitate the aster ejection force during mitosis as a function of chromosome size, mitotic stage and distance from the astral center, and to evaluate whether this force contributes to bipolar attachment and congression. For these studies chromosome arms in NLCs will be severed from the kinetochore region with a laser microbeam and manipulated by calibrated optical tweezers. Aim #4 is to determine, by same-cell V-LM/EM methods, the relationship between the number of MTs at sister PtK(1) kinetochores and the direction the congressing chromosome is moving--information that is critical for distinguishing between hypotheses of congression. Aim #5 is to generate high-resolution 3D models of kinetochore structure, prior to and after the acquisition of MTs, by EM tomographic methods. Such reconstructions are needed to evaluate existing models of kinetochore structure and function. Aim#6 is to determine if poleward chromosome velocity is regulated by kinetochore fiber maturity or the stage of mitosis. This study, which also addresses whether the mechanism for chromosome motion differs between mitotic stages, will be based on V-LM analyses of unattached chromosomes induced to attach to the spindle during anaphase. Finally, Aim #7 is to determine whether metaphase spindle structure is dependent on bipolar-oriented chromosomes, and if the force-producing mechanism for anaphase spindle elongation is active throughout metaphase or triggered only during anaphase. To answer these questions metaphase PtK(1) cells will be followed by V-LM after one of the kinetochores on every chromosome has been destroyed by laser ablation. Together these aims will significantly enhance our concept of how mitosis works--a requisite for understanding the etiology of various birth defects and cancers, and for designing new therapeutic strategies for the control of cell proliferation related to disease states.